The molecular mechanisms involved in ethanol induced tolerance and dependence have yet to be elucidated. The major goal of the applicant's laboratory is to define the mechanisms by which drugs modulate GABA-A- ergic transmission, and to study the molecular mechanisms underlying the development of tolerance to drugs that facilitate GABA-A-ergic transmission in the CNS. The present proposal is an extension of ongoing studies aimed at understanding and further defining the role of GABA-A receptors in chronic ethanol induced tolerance/dependence. The present proposal will test the hypothesis that chronic ethanol treatment changes the sensitivity of the "neurosteroid" site of the GABA-A receptor complex, which may be associated with the development of tolerance/dependence. This will be achieved by using a comprehensive approach including radioligand binding, 36Cl- influx, patch clamp techniques, measurement of GABA-A receptor subunit polypeptide levels by Western blot and immunoprecipitation studies. The focus on the "neurosteroid" site is derived from recent studies suggesting potential involvement of this site in ethanol dependence. The experiments will be performed in well characterized cortical neurons in the absence of pharmacokinetic variability. We considered the intrinsic heterogeneity of the cortical neurons a plus, since it represents diversity of cell types in intact CNS, and we can study binding, function, and protein expression under defined conditions. The aims of the proposal are: I) Does chronic ethanol treatment alter the ability of the neurosteroid, 5alpha-pregnane-3alpha- 01-20-one (5alpha3alpha) to affect the binding of ligands associated with the GABA-A receptor complex. This will be achieved by comparing the effects of 5alpha3alpha on [3H]GABA, [3H] flunitrazepam, and [35S]t- butylbicyclophosphorothionate (TBPS) binding in control, chronic, and withdrawn group of neurons. II) Does chronic ethanol treatment alter the potency and/or efficacy of the neurosteroids to enhance GABA-A-ergic transmission. This will be achieved by examining the effects of 5alpha3alpha on GABA-induced [36Cl-] influx, and GABA-induced whole-cell currents by patch clamp techniques in control, chronic, and withdrawn group of cortical neurons. III) Does chronic ethanol treatment alter the GABA-A receptor subunit polypeptide expression, which may explain the altered sensitivity of the neurosteroids, following chronic ethanol treatment. This will be achieved by using Western blot and immunoprecipitation studies. Our studies will determine the molecular mechanisms which may he associated with enhanced sensitivity of the neurosteroid site, and would result in a facilitatory effect on GABA-A- ergic transmission, that could explain the behavior, i.e. tolerance.